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Use of aqueous two‐phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports
Author(s) -
HernandezJustiz Odette,
FernandezLafuente Roberto,
Terreni Marco,
Guisan Jose M.
Publication year - 1998
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19980705)59:1<73::aid-bit10>3.0.co;2-3
Subject(s) - penicillin amidase , chemistry , immobilized enzyme , ammonium sulfate , hydrolysis , aqueous solution , chromatography , biocatalysis , extraction (chemistry) , aqueous two phase system , peg ratio , yield (engineering) , organic chemistry , enzyme , catalysis , ionic liquid , materials science , finance , economics , metallurgy
Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two‐phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre‐equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients ( K ) of cephalexin in very different aqueous two‐phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600–3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre‐incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 m M phenylglycine methyl ester and 100 m M 7‐amino desacetoxicephalosporanic acid (7‐ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:73–79, 1998.